Project Summary/Abstract: HIV-1 broadly neutralizing antibodies (bnAbs) are difficult to induce with vaccines, but are generated in approximately half of HIV-1-infected individuals after years of chronic infection. Definition of the mechanisms that enable bnAb development during infection will pave the way for vaccination strategies capable of eliciting bnAbs. Some reasons bnAbs are not readily induced are their requirement for high immunoglobulin gene somatic hypermutation that requires successive rounds of B cell affinity maturation in germinal centers and their limitation by host regulatory controls. We have recently identified that HIV-1-infected individuals who developed bnAbs have more dysfunctional natural killer (NK) cells as well as higher frequencies of circulating CD4+ T follicular helper (Tfh) cells that provide critical help to B cells. We further demonstrated that activated NK cells from HIV-seronegative donors could reduce Tfh cell numbers and decrease B cell activity in NK-Tfh- B cell co-cultures. These data led to the scientific premise of our proposal that NK cell dysregulation results in reduced NK cell constraints on Tfh availability which contributes to bnAb development during HIV-1 infection. This identified NK cells as a potential key regulator of the HIV-1 bnAb response. The overall objective of this proposal is to test our central hypothesis that transient blockade of NK cell immunoregulatory activity will create an immunological environment permissive for the development of HIV-1 bnAbs by increasing germinal center T and B cell responses. This hypothesis is supported by results from a preliminary study we have performed in rhesus macaques to address the effects of transient NK depletion on the response to HIV Envelope vaccination, where an increase in germinal center Tfh and B cell frequencies was observed in macaques that had NK cells depleted during HIV vaccination compared with HIV vaccinated animals without NK cell depletion. Our specific aims will test the following hypotheses: (Aim 1) NK cells from HIV-1-infected individuals generating bnAbs will have a more impaired immunoregulatory capacity than those from HIV- infected individuals without bnAbs; (Aim 2) We will define the critical NK cell receptor-Tfh ligands important for NK cell inhibition of Tfh and B cells; (Aim 3) Determine the mechanisms of transient NK cell depletion on the germinal center HIV-1 neutralizing antibody response. This project is innovative because we have identified that NK cell dysfunction contributes to HIV-1 bnAb development and have demonstrated in depletion of NK cells improves germinal center activity after immunization. This contribution is significant because identifying the molecular mechanisms and receptor-ligand interactions that mediate NK cell inhibition of Tfh and B cell responses and determining the effect of NK cell depletion or receptor blockade on germinal center B cell a Tfh cell repertoire and function in vivo will identify novel NK cell-targeting strategies that can be applied during HIV-1 vaccination to improve immune responses.